The lab studies the quantitative operation of the systems that living cells use to sense, represent, transmit, and act upon information to make decisions that determine their future fates. One system under study is a prototypic cell signaling system in budding yeast, the pheromone response system. When appropriate (which is frequently) experimental work proceeds in concert with efforts to account for observed quantitative behaviors by simulation. Lab has extended similar work to systems operating in single cells of tissues in a metazoan, Caenorhabditis elegans. Because the lab studies system operation in single cells, we also study the causes and consequences of cell-to-cell variation in function of these systems, and the still-mysterious persistent cell physiological states that underlie much of this variation. Differences in these physiological states can have significant effects on the function of the organism that continue over the organism's life.
Work requires continual development and refinement of experimental and computational methods. One area of continual development is rapid means to generate new DNA constructions and make desired changes to the genomes of yeast and higher cells. Another is development of intracellular reporters that can quantify particular molecular events in living cells, together with microscopic, flow cytometric, and computational means to read the output of these reporters. Another is fluidic means to provide to the systems defined inputs. Much of this technology development finds application to other biological problems. Some of the current work is suggesting modalities for experimental manipulations and therapeutic interventions. Much of this technology development is in concert with closely collaborating labs in the US and abroad. The suggestions for possible therapeutic intervention may lead to collaborative applied work in 2015.
For the past three years, the lab has also included an experimental social science component. Some of this work continues under the aegis of the Center for Biological Futures, a two-year pilot project that brought together biologists with scholars in the social sciences and humanities, including anthropologists and philosophers, to better understand how biological knowledge and capability are shaping human affairs in the 21st century. This work included a significant collaboration with investigators at the University of Washington, in the project Biological Futures in a Globalized World. Brent and other lab members are frequently able to participate in government and other advisorial settings to help shape the overall course of future research, and all lab members are encouraged to identify and analyze how the outcomes of their research and the ongoing increases in biological knowledge and capability might shape human affairs.
Much of the research at the Hutch and elsewhere relies on new DNA constructions. Since the early 1970s, researchers have continued to use "classical" means to construct DNA molecules, but they have also developed additional powerful means to make complex molecules of desired sequence. In many cases, researchers rely on kits made by commercial manufacturers that use methods the researchers do not understand. A forthcoming paper Current Protocols in Molecular Biology by Bryan Sands and Roger Brent describes and explains the most important methods, including the improved assembly methods based on recombination in yeast developed here.
Derek M. Britain, the most junior lab member, will be graduating from the lab to go enter graduate school in Biophysics at UCSF. Derek joined the lab as an undergraduate in the UW bioengineering program and then stayed on as a full time researcher. Derek's work here was on the response of single yeast cells to mating pheromone. He demonstrated effects of particular allelic variants of microtubule plus end binding proteins on cell-to-cell variation in signal transmitted through the system. The work may be important because these variants occur in homologs of these proteins found in members of the human population, and so may contribute to human disease.
Allen Northrup, a distinguished microfluidics engineer and successful biotech executive,has joined the lab as a visiting scientist. Allen (see bio) is one of the pioneers of microfluidics and among other things is responsible for the first devices that carried out PCR in micromachined silicon. He plans work that may improve the ability of researchers and diagnosticians to detect and quantify rare molecules in dirty samples.
At UCLA, Alexander Mendenhall, now out of stealth mode, presented his results on single cell quantification at the annual worm meeting. Results included studies of effects of configuration of different reporter gene elements total measured fluorescence. This work is important because it provides the necessary foundation for the lab's ongoing studies of variation and physiological state.
A paper by Alexander Mendenhall and other lab members is now published in PLOS ONE. The paper tells researchers how to quantify with great accuracy reporter gene expression in living single cells in adult Caenorhabditis elegans. Lab researchers view development of these quantification methods as the foundation on which much future work worldwide will depend. The work opens the way to understanding the origin of non-genetic variation in living animals-- the "neither" in the triad sometimes termed "nature, nurture, or neither". It will also allow study of now-mysterious physiological states that can affect lifespan and other outcomes.
In work with the lab, Roufan Cao and Jessica Perea Houston at NMSU have shown that phasor plots can distinguish populations of cells in flow cytometry based on fluorescent lifetime. Experiments continue.
More than 10 years ago, Ty Thomson, then a graduate student with collaborator Drew Endy, was working with the lab on the Alpha project, an effort to make a comprehensive predictive model of the yeast pheromone response system. For his PhD thesis (http://dspace.mit.edu/handle/1721.1/45206), Ty carried out a full review of the literature, extracted key facts and parameters from the literature, and documented this in a wiki. Significantly, each fact in the wiki was synonymously encoded in a particular language, BioNetGen, which facilitated its automatic incorporation into an SBML coded model on demand. By this means, changes in the prose wiki automatically rippled through to changes in the model.
Today, eight years after launch, the model site continues to be near the top on searches for quantitative information about the pheromone response system and supply starting materials for researchers modeling cell signaling systems.
Work from the lab in 2008 (Yu et al.) refocused our interest on the fact that some signaling systems turn on downstream events in proportion to the number receptors activated at the cell surface. This is "Dose-Response Alignment", or DoRA. Ongoing work theoretical work by Steve Andrews and other lab members now shows that simple negative feedback is not able to maintain DoRA but that augmented negative feedback and a second control mechanism can achieve it. Experimental work to establish the existence of the second mechanism continues.
In collaboration with Jessica Perea Houston and her coworkers at New Mexico State University in Las Cruces, Bryan Sands, Bill Peria and other lab members have sorted engineered yeast cells engineered to express fluorescent proteins with different lifetimes. The work required Sands to build proteins with different lifetimes and Peria to find and deploy an relatively obscure algorithmic method to program the sorter's Field Programmable Gate Array (FGPA) to compute lifetimes quickly enough to allow sorting. One consequence of this work will be to separate populations of cells in different physiological states defined by differences in cell signaling.
Goertzel, G. (1958) An Algorithm for the Evaluation of Finite Trigonometric Series. American Mathematical Monthly 65 (1): 34–35, doi:10.2307/2310304
25 July 2014
Collaborating PI Jessica P. Houston receives a basic research award from The NCI’s Center to Reduce Cancer Health Disparities (CRCHD) at an investigators workshop for "Partnerships to Advance Cancer Health Equity" (PACHE) for collaborative work with Brent lab. See http://crchd.cancer.gov/news/spotlights/program-spotlights_PACHE_2014.html
1 August 2014
Alexandra Ventura et al. " Utilization of extracellular information before equilibrium receptor binding expands and shifts the input dynamic range", accepted for PNAS. Work is in collaboration with the lab of Alejandro Colman-Lerner lab at the University of Buenos Aires. The mechanism, PRESS, is important for the operation of the large number of cell signaling systems that share certain dynamic properties.
5 September 2014
Bryan Sands et al, "Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes" is accepted in PLOS ONE. Work is in collaboration with the lab of Jessica P. Houston at New Mexico State University. Going forward, we hope that altered-lifetime fluorescent proteins and appropriate equipment will enable us to quantify signaling events involving very small (<10) numbers of molecules.
23 September 2014
Eddie Altszyler et al. "Impact of upstream and downstream constraints on a signaling module’s ultrasensitivity " is accepted in Physical Biology. Work is from collaborating Colman-Lerner lab. If certain "modules" in cell signaling systems generate steep (or "switchlike") response curves, and interface with certain kinds of upstream or downstream reactions, the "modules" can give response curves that are even steeper.